Peptides and remedy for bone diseases containing the same as active ingredient

ABSTRACT

A new substance useful as a drug for the treatment of osteopathy is disclosed. The present invention provides a peptide and derivative thereof, the peptide having an amino acid sequence SEQ ID NO: 1 designated hereunder as follows, wherein both of the peptide and derivative thereof having growth promotion effects and activity promotion effects on osteoblasts, as well as a drug having the peptide as an effective component for the treatment of osteopathy: 
     
       
         
               
             
                 Lys Leu Thr Thr Ile Phe Pro Leu Asn SEQ ID NO: 1. 
               
                  1               5 
               
                   
               
                 Trp Lys Tyr Arg Lys Ala Leu 
               
                  10                 15

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/930,776, filed on Jan.4, 1999, now U.S. Pat. No. 6,071,881 entitled, “Novel Peptides andRemedy for Bone Diseases Containing the Same as Active Ingredient”,which was the National State of International Application No.PCT/JP96/00915, filed Apr. 3, 1999.

FIELD OF THE INVENTION

The present invention relates to a new drug containing the same as aneffective component for treating osteopathy.

BACKGROUND OF THE INVENTION

It is well known that there are a number of physiological activesubstances in a human body, and that such substances are closelyinvolved in the support of normal biological activities in a human body.Both of such physiological active substances of a human body andsynthesized physiological active substances are showing possibilities ofbeing new drugs as well as providing new insights into the developmentof new drugs. Therefore, the search for such physiological activesubstances is of utmost importance.

On the other hand, for the treatment of osteopathy such as osteoporosis,calcitonin, female hormone, and activated vitamin D3 and the like arecurrently used, the remedial effects thereof are not necessarily ofsatisfactory one.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a new substance whichis useful as a drug for the remedy of osteopathy.

The present inventors found, while conducting various experiments tosearch for a substance which promotes the activities of osteoblasts, anew peptide having foregoing effects on osteoblasts which is involved inthe bone formation, and the present invention was made through such afinding.

In other words, the present invention provides a peptide and aderivative thereof, said peptide having an amino acid sequencedesignated by the sequence number 1 shown in the sequence table attachedherewith, wherein both of said peptide and said derivative thereofhaving growth promotion effects and activity promotion effects onosteoblasts. In another aspect of the present invention, it provides adrug having said peptide of the present invention as an effectivecomponent for treating osteopathy.

The present invention is further explained in detail hereunder.

BEST MODE FOR CARRYING OUT THE INVENTION

The peptide of the present invention has basically an amino acidsequence designated by sequence number 1 shown in the sequence tableattached herewith.

Furthermore, with the scope of the present invention, in addition tosaid peptide having an amino acid sequence designated by the sequencenumber 1 shown in the sequence table attached herewith, a derivative ofsaid peptide is included, wherein said derivative having growthpromotion effects and activity promotion effects on osteoblasts. Forexample, any “derivative peptide” referred to in the present inventionmay include any derivative of said peptide obtained by any chemicalmodification thereof which may not impede any of said effects. Forexample, it is well known that by replacing the carboxyl radical at theC end of a peptide with an amino radical, the stability thereof in ahuman body is increased, and any of such chemically modified peptide isincluded in the scope of the present invention.

Still further, it is well known to anyone skilled in the art that, incase of a peptide which generally has physiological activity, even whensome of the amino acids thereof are replaced by other amino acids, whensome amino acids are added thereto, or when some of the amino acids aredeleted therefrom, there are cases where the physiological activity ismaintained. Therefore, in case of the scope of the present invention,the “derivative peptide” includes any peptide wherein some of the aminoacids which form said peptide having an amino acid sequence designatedby the sequence number 1 are replaced by some other amino acids, whereinsome amino acids are added thereto, or wherein there are some aminoacids deleted therefrom, and furthermore, among such derivativepeptides, any peptide having growth promotion effects and activitypromotion effects on osteoblasts are included in the scope of thepresent invention. For example, the scope of the present inventionincludes a peptide where not more than 5 amino acids are added, replacedor deleted, or a peptide wherein the degree of homology of the aminoacid sequence is not less than 70% and said peptide having growthpromotion effects and activity promotion effects on osteoblasts.Furthermore, whether a derivative of a peptide has growth promotioneffects and activity promotion effects on osteoblasts may be examined bythe method of Examples 2 and 4 described hereunder.

The peptide of the present invention designated by the sequence number 1has a rather small number of amino acid components, with such numberbeing 16, it may be easily synthesized by a chemical synthesizing methodof any known method. For example, it may be easily produced by using acommercially available peptide synthesizing machine. It may also beeasily produced by any of known generic engineering methods, wherein aDNA encoding said amino acid sequence is synthesized by using a DNAsynthesizing machine, followed by inserting said DNA into the cloningportion of a commercially available cloning vector thereby transformingthe host microorganism, followed further by culturing the same.

As has been experimentally confirmed in the examples describedhereunder, the peptide of the present invention has growth promotioneffects and activity promotion effects on osteoblasts. Therefore, thepeptide of the present invention is useful as a drug for the treatmentof osteopathy such as osteoporosis. Osteopathy which can be cured by thepeptide of the present invention may be found, for example, inosteomalacia and the like other than osteoporosis.

Because the molecular weight of the peptide of the present invention isrelatively low, it may be intravenously administered, hypodermicallyadminis- tered, intramuscularly administered, as well as orallyadministered or percutaneously administered after the dosage is preparedby any of the known method.

Although the amount of dosage may be appropriately decided depending onthe patient's condition, it may be generally in the order of 0.1 to 10mg of the peptide of the present invention per an adult per each day.

Furthermore, in case of intravenous administration, hypodermicadministration or intramuscular administration, the peptide of thepresent invention is preferably administered after dissolving the sameinto a weak acid buffer solution such as a citric buffer solution (pH4˜6) or acetic buffer solution (pH 4˜6). In such a case, theconcentration of the peptide in the buffer solution is generally in theorder of 0. 1 mg/ml ˜10 mg/ml. In case of oral administration orpercutaneous administration, the peptide is preferably dissolved into afat-soluble substance (for example, petrolatum and the like) tofacilitate the increase of absorption characteristics. In such a case,the concentration of the peptide of the present invention is generallyin the order of from 0.1 mg/ml to 100 mg/ml.

EXAMPLES

The present invention is further described in detail with reference toexamples. However, the scope of the present invention is not restrictedto the examples described hereunder.

Example 1

A peptide having an amino acid sequence designated by the sequencenumber 1 was synthesized by using a commercially available peptidesynthesizing machine.

Example 2 Effects on Growth Promotion of Osteoblasts

ROS cells, which are osteoblasts, obtained from a rat (source: ATCC) arecultured on a F10 medium containing 10% of the serum of a calf embryo,followed by a growing in a thermostat being set at 37° C. and under ahumidification by 5% carbon dioxide gas. Seeding of 1×105 cells/well ona culture plate with 24 wells was effectuated after trypsinization,followed by replacing said medium with a F10 medium having 1% of theserum of a calf embryo when the former became confluent, and thereafterit was cultured for 24 hours. Then, the peptide of the present inventionproduced in Example 1 of this invention was dissolved in a F10 mediumhaving 1% of the serum of a calf embryo, and it was added to wells whilethe amount thereof being varied, followed by continuation of the cultureprocess for another 24 hours. After the culture process, the cell growthpromotion effects of the present peptide was measured by MTT assay toobtain the growth promotion effects in comparison with the untreatedgroup. MTT assay and the calculation of the rate of growth promotionwas, in effect, carried out as follows. According to the procedure ofMTF-Cell-Growth Assay kit commercially available from Funakoshi Co.,Ltd., after adding the present substance to the wells while the amountthereof being varied and followed by leaving the same for a whole day,the number of living cells were counted by colorimetric analysis byusing a phenomenon wherein MTT (3-4, 5-Dimethylthiazol-2-YL)-2,5-Diphenyl Tetrazolium Bromide is cleaved to produce formazan havingdark blue color by an enzyme present in the mitocondrion of a livingcell. The colorimetric results of the group obtained by adding varyingamount of the present substance thereto, while the control group towhich none of the present substance was added being as 100%, are shownas follows. The results are shown in the following Table 1.

TABLE 1 Amount of Peptide Added Rate of Growth Promotion (μg/well) (%) 0100.0 0.001 109.6 0.01 110.5 0.1 636.2 1.0 1317.1

As can be seen from Table 1, it was confirmed that the peptide of thepresent invention provides growth promotion effects on osteoblasts.Therefore, it is considered that the peptide of the present invention isrelated to an increase of the amount of bone, and it is useful for thetreatment of osteopathy such as osteoporosis and the like.

Example 3 Existence of a Receptor for the Peptide of the PresentInvention on an Osteoblast

Because it was found that the peptide of the present invention hasgrowth promotion effects on osteoblasts, it can be inferred thatosteoblasts have receptors for the present peptides. If there is anyreceptor, the present peptide can be considered to be the fundamentalsubstance for the living organisms or cells, and further analysis wascarried out to find out whether the osteoblast has any receptor or not.

The peptide obtained in Example 1 was marked by biotin, and then, thepresent peptide after being marked to a certain degree was added to ROScells cultured in the same way as that of Example 2, wherein the presentpeptide without any marking was dissolved, to effectuate competingreaction, into a F10 medium having 10% of the serum of a calf embryo,followed by adding the same, while varying the amount thereof, to saidcells to see the competing reaction. The actual process of thisexperiment was as follows. According to the procedures of a proteinbionizing and labeling kit available from UMILON, the present peptidewas biotinized, and then, a certain amount of biotinized peptide wasadded to a certain number of cells seeded in wells, then from 0 to 0.512μg/well of unlabeled peptide was added to each cell to effectuate thecompeting reaction for 6 hours, and thereafter, the cell was washed byPBS and then the coloration reaction, which was caused by peroxidaselabeled with streptoabidin reacting with biotinized peptide bonded toreceptors present on the cell surface, was observed. When any receptorfor the present peptide is present on the cell surface, there is areaction competing with the unlabeled peptide whereby the colorationdegree is decreased. The results are shown in Table 2 hereunder.

TABLE 2 Amount of Unlabeled Ratio to the Peptide Added (μg/well) LabeledPeptide (%) 0 100 0.032 98.4 0.064 86.5 0.128 79.6 0.256 34.1 0.512 29.5

As shown in Table 2 above, because the ratio to the added labeledpeptide varies depending on the amount of unlabeled peptide, it isobvious that the osteoblast has receptors for the present peptide, andit is further inferred that the present substance has a fundamentalrole.

Tests on Acute Toxicity

By using the peptide prepared in Example 1, its acute toxicity wastested on ddy male mice (with the body weight ranging from 40 to 45 g).The present peptide was dissolved in a physiological saline (pH 6.0) andthe solution thereof was administered intravenously into a vein in thetail of each mouse, and observation was made for 14 days. The dosageamount was set respectively at 1, 10, and 100 μg/kg. The results areshown in Table 3 hereunder.

TABLE 3 Dosage Amount (μg/kg) Number of Deceased Mice 1 0/5 10 0/5 1000/5

As can be seen from the above table, there was not deceased mouseobserved up to the dosage amount of 100 μg/kg.

Industrial Applicability

A new peptide is provided by the present invention. The peptide of thepresent invention has, as has been experimentally confirmed by the aboveexamples, growth promotion effects and activity promotion effects onosteoblasts, and it has been made obvious that such peptide has effectsas a drug for the treatment of osteopathy.

Sequence Table

Sequence Number: 1

Sequence Length: 16

Type of Sequence: Amino Acid Sequence:

Lys Leu Thr Thr Ile Phe Pro Leu Asn Trp Lys Tyr Arg Lys Ala Leu

1 16 Amino Acids Amino Acid Unknown Peptide No No Entire Peptide PeptideSynthesizer Peptide Synthesizer Promotion effects on osteoblasts 1 LysLeu Thr Thr Ile Phe Pro Leu Asn Trp Lys Tyr Arg Lys Ala 1 5 10 15 Leu

What is claimed is:
 1. A peptide consisting of at least 12 and no morethan 21 amino acid residues in length and having at least 70% homologyto the sequence of sequence SEQ ID NO:1, wherein said peptide has growthpromotion effects and activity promotion effects on osteoblasts.
 2. Apeptide having no more than five amino acid residues which are differentthan the peptide of SEQ ID NO:1, wherein said peptide has growthpromotion effects and activity promotion effects on osteoblasts.
 3. Apeptide consisting of the amino acid sequence of SEQ ID NO:1, whereinone or more of the amino acids residues have been chemically modified,wherein said peptide has growth promotion effects and activity promotioneffects on osteoblasts.
 4. A peptide of claim 3, wherein the chemicalmodification is the replacement of the carboxyl radical at the Cterminus of the peptide with an amino radical.
 5. A peptide of claim 3,wherein the chemical modification is to one or more of the amino acidsside chains.